Efficient formation of inert Bi-213 chelates by tetraphosphorus acid analogues of DOTA: towards improved alpha-therapeutics

Background The recently growing interest in targeted alpha-therapy (TAT) calls for improvement of the labelling chemistry of the corresponding radionuclides. 213BiIII is a short-lived alpha emitter which emits only one alpha particle in its decay chain. Hence, it might be safer in application than other respective nuclides, such as 223Ra or 225Ac, because no alpha-emitting daughters are released upon recoil. We investigated cyclen derivatives with phosphorus-containing pendant arms regarding their suitability for 213Bi labelling. Results The concentration dependency of 213Bi labelling at 25 °C and 95 °C was determined for DOTP, DOTPH, DOTPEt, and DOTPI, as well as for DOTA and CHX-A"-DTPA for comparison. The labelling efficiency of the phosphorus-containing ligands was at least comparable to CHX-A"-DTPA and exceeded that of DOTA. DOTP was most efficient, requiring chelator concentrations for labelling which were approx. two orders of magnitude lower than those required for CHX-A"-DTPA, both at 25 °C and 95 °C. The 213Bi complexes of phosphorus ligands furthermore showed a higher stability against demetallation (> 96% of intact complex after 120-min incubation in plasma were found for DOTP, DOTPH, and DOTPEt, compared to 85% for DOTA and 76% for CHX-A"-DTPA). Conclusion Cyclen derivatives bearing four N-methylenephosphonic or -phosphinic acid substituents, e.g., DOTP, are capable of complexing the alpha-emitting radionuclide 213BiIII with higher efficiency and in-vitro stability than the current gold standards DOTA and CHX-A"-DTPA.


Background
Compared to βor γ-radiation, tissue interaction of α-particles is characterized by a higher linear energy transfer (LET) and a much higher cell toxicity due to an enhanced probability of causing DNA double strand breaks [1]. In addition, the low tissue penetration depth of α-radiation (3-4 cell diameters) entails a more localized therapeutic effect, ideal for killing remaining single cancer cells or micrometastases which, in most conventional treatment regimes, can survive and later function as nuclei of tumor recurrence. Nevertheless, radionuclide therapy of cancer using radiopharmaceuticals labeled with α-emitting radionuclides (referred to as "targeted alpha therapy," TAT) has hitherto played only a limited clinical role, although the therapeutic potential of the α-emitter 225 Ac (T ½ = 9.92 d) [2] has been emphasized already in 2001 [3]. The recent approval and market entry of 223 Ra chloride as an α-emitting therapeutic radiopharmaceutical [4] and successful application of 225 Ac-labeled inhibitors of prostate-specific membrane antigen (PSMA) for treatment of prostate cancer [5] highlighted the clinical potential of α-therapy and led to a tremendous boost of attention for TAT.
However, despite of proven suitability of 225 Ac for treatment of terminal cases, such as β-refractory prostate cancer patients [5], there are still concerns regarding safe applicability of this nuclide for other than palliative use. There are four α-decays in its multistep decay scheme, while the recoil of the first α-emission releases the nuclide from the binding site (typically a chelate) [6]. In adverse cases, slow or incomplete internalization into cells may lead to uncontrolled distribution of the various α-emitting daughter nuclides in the body, causing the risk of severe side effects (e.g., kidney toxicity or carcinogenesis) owing to undesirable irradiation of healthy tissue [7]. In view of these issues, 213 Bi (T ½ = 46 min) [8], a late daughter nuclide of 225 Ac, appears to be a valuable alternative. Diffusion after recoil is not a problem because a stable isotope, 209 Bi, is obtained after decay via nearly simultaneous αand β-emissions, while an additional 440 keV γ-line enables scintigraphic imaging. 213 Bi is conveniently obtained from 225 Ac/ 213 Bi generators, small shielded chromatographic benchtop devices containing 225 Ac III adsorbed to an organic matrix, from which 213 Bi III is eluted with iodide solution in form of the [ 213 BiI 4 ] − and [ 213 BiI 5 ] 2− complexes [9]. Hence, 213 Bi has been exploited for various therapeutic applications [10][11][12], none of which, however, reached clinical routine so far, above all, due to very limited availability of 213 Bi. Nevertheless, the awakened interest in 225 Ac and the foreseeable expansion of global 225 Ac production capacity will also entail a wide availability of 213 Bi generators in the near future [13]. Overall, a higher inherent safety of 213 Bi, resulting from its short half-life and a decay scheme involving only a single α-decay, renders this nuclide attractive for future development of α-therapeuticals which, in turn, calls for improvement of the corresponding labelling chemistry that hitherto received only little attention.
Radio-TLC was performed on glass microfiber chromatography papers impregnated with silica (ITLC®, Agilent). Using 0.1 M aq. sodium citrate as eluent, non-incorporated 213 Bi III is transformed into the citrate complex which moves with the solvent front, while all chelates are sufficiently retained to enable ground-line separation (R f < 0.5). Readout of chromatograms was done using a BIOSCAN TLC scanner, consisting of B-MS-1000 scanner, and B-EC-1000 detector with a B-FC-3600 GM tube. 213 [16]. The eluate was adjusted to pH 5.5 with 1 M aq. NaOAc buffer (1.6 mL). Labelling was performed by addition of the buffered eluate (90 μL) into an Eppendorf cup containing the precursor solution (10 nM-1 mM, 10 μL), resulting in final chelator concentrations of 0.001-100 μM. After 5 min of incubation at ambient temperature (approx. 25°C) or at 95°C, the fraction of complexed 213 Bi III was evaluated by radio-TLC.

Stability studies
Stability of 213 Bi III -complexes was tested in human plasma or 0.1 M aq. Na-DTPA (pH 7.5) by addition of 10 μL of the labelling solution (with 1 mM ligand concentration), containing the radiometal complex, to 90 μL of the

Results
Up to now, 213 Bi III labelling virtually exclusively relied on well-established acyclic or cyclic polyamino-polycarboxylate ligands, above all, CHX-A"-DTPA [17] or the highly popular and versatile chelator DOTA [18] (Fig. 1). However, we previously noticed that 1,4,7-triazacyclononanes bearing phosphinic acids as N-substituents (TRAP chelators) [19] show superior labelling efficiency for the short-lived trivalent positron emitter 68 Ga III [20,21] in comparison to their parent tricarboxylate NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) [22], pointing at potentially superior radiolabelling properties of phosphorus-pendant azamacrocycles in general. Thus, we elucidated the potential of cyclen-based chelators with phosphorus-based N-pendant arm donors for 213 Bi III complexation. For this purpose, 213 Bi III labelling of phosphonic acid derivatives DOTP [23] and DOTP OEt [24] as well as of phosphinic acids DOTP H [25] and DOTPI [14] was compared to the aforementioned standard scaffolds DOTA [26] and CHX-A"-DTPA [27], also under mild conditions (ambient temperature, pH 5.5) compatible with any type of biological targeting vector, including antibodies ( Fig. 2; Tables 1 and   2). DOTA shows the poorest performance among all chelators investigated and, as with virtually all other radiometals, apparently cannot be labeled quantitatively with 213 Bi within reasonable ranges of concentration and time at ambient temperature. This is why open-chain chelators, particularly CHX-DTPA derivatives, are usually applied for this purpose, despite of inherently lower in vivo stability of their M III complexes [28]. However, to our surprise, the performance of phosphorus-based cyclens was found at least comparable to CHX-A"-DTPA, while DOTP showed particularly efficient radiolabelling, most likely due to higher affinity of trivalent bismuth to the relatively hard phosphonate oxygen atoms. This is a remarkable finding because in contrast to open-chain ligands, metal ion complexation by cyclic chelators usually occurs slower, via a two-step mechanism [29]. An initially formed out-of-cage complex, wherein the metal ion is coordinating only to side arm oxygen donors and solvent (water) molecules [25] is transformed into the in-cage complex, characterized by a N 4 O 4 coordination mode of the ligand, via a substantial energy barrier. In terms of radiolabelling, this barrier causes a slower activity incorporation, but, on the other hand, is also related to higher kinetic inertness of the radiometal complexes which translates to lower dissociation rates.
To assess this important parameter, we characterized the stability of the 213 Bi chelates in a transchelation challenge against DTPA, and in human plasma at 37°C. Figure 3 and Table 3 show that in accordance with expectations, the 213 Bi III -complex of the open-chain ligand CHX-A"-DTPA exhibits the lowest kinetic inertness, resulting in a larger extent of dissociation than observed for the cyclic systems. Among the latter, all phosphorus ligands show quite similar resistance against demetallation. Notably, their 213 Bi III complexes are also more inert than that of DOTA, most likely because they are protonated at lower a pH [15,30].

Discussion
With a > 90% stability in plasma over the entire dosimetrically relevant time period of 213 Bi (approx. three half-lives), the phosphinate and phosphonate chelators appear better suited for a safe application in 213 Bi therapeutics than CHX-A"-DTPA derivatives. In addition, the higher labelling efficiencies, i.e., lower molar amounts of chelator required For more data see Table 3 Table for the same extent of radiometal incorporation, will provide radiopharmaceuticals with higher specific activity, that is, an improved ratio of labelled vs. non-labelled compound in the final preparation. Hence, by administration of the same amount of, e.g., a 213 Bi labelled antibody, a multiple amount of activity could be deposited in the target (tumor) tissue, resulting in a substantially increased radiation dose per tissue volume and, consequently, in a more successful therapy.

Conclusion
In conclusion, we found that 213 Bi III complexation properties of cyclen-based phosphinate and particularly of phosphonate ligands are superior to the gold standard acyclic or cyclic chelators for 213 Bi III , CHX-A"-DTPA and DOTA, respectively, reaching comparable labelling yields at 2-4 orders of magnitude lower concentrations both at ambient and elevated temperatures. In view of such highly efficient 213 Bi incorporation, the phosphorus chelators appear ideal for application in freeze-dried labelling kits as known from 99m Tc tracers and in antibody conjugates for immunotherapy where they would offer the benefits of improved in-vivo stability and higher target doses due to higher specific activity. Because at last, targeted α-therapy is widely entering clinical healthcare schemes after remaining in an experimental state for decades [13], our results are expected to support the currently increasing efforts towards advanced 213 Bi radiotherapeutics for improved treatment of cancer.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions JS and CS performed the radiochemical work. JN, JS, and PH drafted the manuscript. All authors participated in the study design, revised the manuscript, and approved the final manuscript.
Ethics approval and consent to participate Not applicable.

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